{"created":"2023-05-15T15:23:30.516296+00:00","id":10352,"links":{},"metadata":{"_buckets":{"deposit":"7f17842d-111b-486d-94a5-1ceab0813bb0"},"_deposit":{"created_by":15,"id":"10352","owners":[15],"pid":{"revision_id":0,"type":"depid","value":"10352"},"status":"published"},"_oai":{"id":"oai:sucra.repo.nii.ac.jp:00010352","sets":["94:429:431:432:506"]},"author_link":[],"item_113_alternative_title_1":{"attribute_name":"タイトル（別言語）","attribute_value_mlt":[{"subitem_alternative_title":"哺乳動物におけるN型糖タンパク質の新規分解経路の研究"}]},"item_113_biblio_info_9":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2015","bibliographicIssueDateType":"Issued"}}]},"item_113_date_35":{"attribute_name":"作成日","attribute_value_mlt":[{"subitem_date_issued_datetime":"2015-12-16","subitem_date_issued_type":"Created"}]},"item_113_date_granted_20":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2015-03-24"}]},"item_113_degree_grantor_22":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"埼玉大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"12401","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_113_degree_name_21":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(学術)"}]},"item_113_description_13":{"attribute_name":"形態","attribute_value_mlt":[{"subitem_description":"86 p.","subitem_description_type":"Other"}]},"item_113_description_23":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"In eukaryotes, a large portion of the secretory and membrane proteins that synthesized in the ER are modified with asparagine-linked glycans (N-glycans). The covalent attachment of glycans on proteins results in changes in their physicochemical properties, as well as physiological properties.\nThe molecular details of the biosynthetic pathway of N-glycans in mammals have been well-clarified. Dolichol-linked precursors for N-glycans are assembled at the ER membrane and transferred to Asn residues of the consensus sequence (Asn-Xaa-Ser/Thr, Xaa≠Pro) in nascent polypeptides. The N-glycosylated proteins, or N-glycoproteins that acquired the correct folding state with the aid of various ER lumenal chaperones, are transported to their respective destinations via vesicular trafficking. Within the secretory pathway, the N-glycans are extensively remodeled from high mannose-type glycans to complex-type glycans that often play central roles in regulating bioactivity or stability of glycoproteins. \nIn contrast to the N-glycan biosynthesis, molecular mechanism for N-glycan degradation has not yet been fully understood even in mammalian cells. For instance, it has long been believed that lysosomes are the predominant organelle to break down all kinds of macromolecules including N-glycoproteins. However, recent studies revealed a novel non-lysosomal glycan degradation pathway occurring in the cytosol, whilst the quality control machinery in the ER was being clarified. The ER lumen possesses quality control system that ensures only correctly folded proteins to exit the ER to their destinations, while those misfolded ones would be retrotranslocated to the cytosol for proteasomal degradation. The latter process was often called as ER-associated degradation (ERAD). During the ERAD process, a cytosolic peptide:N-glycanase (PNGase), which cleaves glycans from glycoproteins, initiates the non-lysosomal degradation of N-glycans by releasing free oligosaccharide (fOSs) in the cytosol. The released fOSs are then further processed by the action of cytosolic endo-β-N-acetylglucosaminidase (ENGase) that removes a single GlcNAc residue from the reducing end on the fOSs, as well as the cytosolic α-mannosidase Man2C1 before the remaining fOSs could be transported to the lysosomes for their degradation into monomeric sugars. In addition to this cytosolic glycan degradation, recent study in our lab indicated unknown functions of basal autophagy in the catabolism of complex type free glycans. These findings have shed light on the importance of non-lysosomal compartments in glycan degradation. However, detailed mechanisms underlying the non-conventional glycan degradation pathways remains largely elusive.\nThe purpose of my study is to elucidate the biological importance and molecular mechanisms of these glycan/glycoprotein degradation pathways. In the first chapter, I aim to clarify the physiological role of the cytosolic PNGase which works in the initial step of the non-lysosomal degradation pathway. This study is extremely important because growing number of patients showing multiple symptoms have been identified harboring mutations in NGLY1 (a gene encoding an orthologue of human cytosolic PNGase), while the development of therapeutic treatments is hindered due to the limited knowledge on the underlying mechanisms of the phenotypes caused by the mutation of NGLY1 gene. Gene-knockout mice studies in our lab have revealed that the abrogation of Ngly1 (mouse PNGase) causes embryonic/perinatal lethality. Surprisingly, this lethality was rescued by an extra knockout of a gene encoding the cytosolic ENGase, clearly indicating that the presence of ENGase in Ngly1−/− mouse causes the lethal phenotype. To provide the mechanistic insight into the ENGase-caused lethality in Ngly1−/− mice, I utilized mouse embryonic fibroblast (MEF) cells derived from various knockout mice, and tried to analyze the activity of ENGase on glycoproteins in the Ngly1−/− MEF cells. Using a plant-derived glycoprotein for ERAD substrate, dysregulation of ERAD was shown in Ngly1−/− MEF cells while normal ERAD was observed in wild type, Engase−/−, and Engase−/−Ngly1−/− MEF cells. Moreover, a deglycosylating activity of ENGase toward a model ERAD substrate was confirmed in the absence of Ngly1, and the ENGase-generated N-GlcNAc containing proteins formed stable aggregates in Ngly1−/− MEF cells. Collectively, this study underscores the functional importance of Ngly1 in the ERAD process and provides a potential mechanism underlying the phenotypic consequences of a newly-emerging genetic disorder caused by mutation of human NGLY1 gene.\nIn the second chapter, I aim to clarify how basal autophagy regulates the degradation of complex type free oligosaccharides. Recent observations in our lab showed that sialyloligosaccharides, which are normally generated and degraded in the lysosomes, considerably accumulated in the cytosol of cells lacking Atg5, a molecule essential for autophagosome formation. Sialin, a sialic acid transporter in the lysosome membrane, was suggested to be somehow involved in the accumulation of sialic acid containing fOSs in Atg5−/− MEF cells. To better understand the underlying mechanisms, I examined the level of sialin molecule upon inhibition of autophagy, and the results clearly showed the increase of sialin proteins upon the inactivation of autophagy process, indicating the specific regulation of sialin by the autophagy. While the detailed mechanisms still remain to be clarified, my results clearly showed the relationship between autophagic process and stability of sialin protein.\nThe observations in my thesis study underscore the biological importance of the novel non-lysosomal compartment in glycan degradation, and expanded our current knowledge of the non-conventional glycoprotein degradation pathways.","subitem_description_type":"Abstract"}]},"item_113_description_24":{"attribute_name":"目次","attribute_value_mlt":[{"subitem_description":"ABBREVIATIONS 1\nSUMMARY 3\nINTRODUCTION 7\nChapter 1. Functional studies of the cytosolic deglycosylating enzymes in mammalian\ncells ...............................................................................................................18\nAbstract .........................................................................................................18\nIntroduction ...................................................................................................19\nResults ...........................................................................................................22\nDiscussion .....................................................................................................44\nExperimental Procedures ..............................................................................49\nChapter 2. The involvement of autophagy in the catabolism of cytoslic free glycans\n.......................................................................................................................57\nAbstract .........................................................................................................57\nIntrodction .....................................................................................................58\nResults ...........................................................................................................62\nDiscussion .....................................................................................................68\nExperimental procedures ..............................................................................71\nACKNOWLEDGEMENTS 75\nREFERENCES 78","subitem_description_type":"Other"}]},"item_113_description_25":{"attribute_name":"注記","attribute_value_mlt":[{"subitem_description":"指導教員 : 鈴木匡","subitem_description_type":"Other"}]},"item_113_description_33":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"subitem_description":"text","subitem_description_type":"Other"}]},"item_113_description_34":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_113_dissertation_number_19":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"甲第970号"}]},"item_113_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.24561/00010346","subitem_identifier_reg_type":"JaLC"}]},"item_113_publisher_11":{"attribute_name":"出版者名","attribute_value_mlt":[{"subitem_publisher":"埼玉大学大学院理工学研究科"}]},"item_113_publisher_12":{"attribute_name":"出版者名（別言語）","attribute_value_mlt":[{"subitem_publisher":"Graduate School of Science and Engineering, Saitama University"}]},"item_113_record_name_8":{"attribute_name":"書誌","attribute_value_mlt":[{"subitem_record_name":"博士論文（埼玉大学大学院理工学研究科（博士後期課程））"}]},"item_113_text_3":{"attribute_name":"著者 ローマ字","attribute_value_mlt":[{"subitem_text_value":"HUANG, Chengcheng"}]},"item_113_text_31":{"attribute_name":"版","attribute_value_mlt":[{"subitem_text_value":"[出版社版]"}]},"item_113_text_36":{"attribute_name":"アイテムID","attribute_value_mlt":[{"subitem_text_value":"GD0000633"}]},"item_113_text_4":{"attribute_name":"著者 所属","attribute_value_mlt":[{"subitem_text_value":"埼玉大学大学院理工学研究科（博士後期課程）理工学専攻"}]},"item_113_text_5":{"attribute_name":"著者 所属（別言語）","attribute_value_mlt":[{"subitem_text_value":"Graduate School of Science and Engineering, Saitama University"}]},"item_113_version_type_32":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"黄, 澄澄","creatorNameLang":"ja"},{"creatorName":"ファン, チェンチェン","creatorNameLang":"ja-Kana"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2018-01-23"}],"displaytype":"detail","filename":"GD0000633.pdf","filesize":[{"value":"7.8 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"GD0000633.pdf","objectType":"fulltext","url":"https://sucra.repo.nii.ac.jp/record/10352/files/GD0000633.pdf"},"version_id":"6713704b-c567-435f-9f6e-00e1112840ac"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis","resourceuri":"http://purl.org/coar/resource_type/c_db06"}]},"item_title":"Studies on non-conventional degradation pathway for N-glycoproteins in mammalian cells","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Studies on non-conventional degradation pathway for N-glycoproteins in mammalian cells","subitem_title_language":"en"}]},"item_type_id":"113","owner":"15","path":["506"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2015-12-17"},"publish_date":"2015-12-17","publish_status":"0","recid":"10352","relation_version_is_last":true,"title":["Studies on non-conventional degradation pathway for N-glycoproteins in mammalian cells"],"weko_creator_id":"15","weko_shared_id":-1},"updated":"2023-06-23T04:29:18.541303+00:00"}